Difference between revisions of "Part:BBa K1720003"
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This device with a GFP reporter was then transfected into HEK293 cells by lentiviral vector.Once we silence the PDE5A gene the level of cGMP will be up regulated as a result. The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain scilencing device. We used Elisa to detect cGMP level. The results are as follow: | This device with a GFP reporter was then transfected into HEK293 cells by lentiviral vector.Once we silence the PDE5A gene the level of cGMP will be up regulated as a result. The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain scilencing device. We used Elisa to detect cGMP level. The results are as follow: | ||
| − | [[File:SCUT China shRNA1.png| | + | [[File:SCUT China shRNA1.png|250px|thumb|left|Green fluorescence signal was observed under fluorescence microscope]][[File:SCUT_China_Elisa_of_scilencing__device.png|250px|thumb|left|cGMP concentration after treat with differenct scilencing device]][[File:SCUT_China_Elisa_of_SNP.png|250px|thumb|left|cGMP concentration after treat with 10umol/L SNP for different time gradient]] |
Revision as of 12:24, 5 September 2015
Human phosphodiesterase 5A gene silencing device NO.1
This device is uesd for silencing the human phosphodiesterase 5A (PDE5A) gene.A U6 promoter driving a designed, synthetic shRNA-like miRNA followed by the terminator.
PDE5A is a cGMP-binding, cGMP-specific phosphodiesterase, a member of the cyclic nucleotide phosphodiesterase family. This phosphodiesterase specifically hydrolyzes cGMP to 5'-GMP. It is involved in the regulation of intracellular concentrations of cyclic nucleotides and is important for smooth muscle relaxation in the cardiovascular system.
We designed 3 silencing device and test their function at the same time.Here is the another two device :BBa_K1720004,BBaK172005
This device with a GFP reporter was then transfected into HEK293 cells by lentiviral vector.Once we silence the PDE5A gene the level of cGMP will be up regulated as a result. The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain scilencing device. We used Elisa to detect cGMP level. The results are as follow:
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 273
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 247
- 1000COMPATIBLE WITH RFC[1000]
Vector Map：
Vector Components:
Virus Titer: (3.23±2)×10^8 TU/ml
Funtional titer is determined based on q-PCR amplification of a small fragment from the lentiviral vector-WRPE that is integrated into the genome of transduced 293T cells.
Experiment 1:
At the beginning of our experiment, we aimed to prove that HEK293 cells can be transfected by our vector. In our vector we inserted EGFP gene as a repoter.Once HEK293 cells are transfected successfully green fluorescence signal will be observed under fluorescence microscope.
Protocol： 1. Seed cells to be 40% confluent at a 35mm culture dish.
2. Dilute 10ul lentiviral vector in 1ml DMEM medium containing 10% FBS
3. Withdraw culture medium from 35mm culture dish.
4. Add vector-DMEM complex to cells
5. Incubate for 15 hours.
6. Withdraw vector-DMEM complex from culture dish.
7. Add 2ml DMEM medium containing 10% FBS to cells and incubate for 10 hours
8. Observe the cells under Inverted fluorescence microscope.
Result:
From the picture we can see that vivo green fluorescence signal was observed which indicated that HEK293 cells had been transfected successfully!
Experiment 2:
After we proved that HEK293 cells can be transfected, PDE5A gene expression levels were determined by real-time PCR.
Protocol：
Result:
Experiment 3:
After we scilencing the PDE5A gene,we used cGMP Elisa kit to detect the cGMP concentration to see whether cGMP concentration can be up regulated by our scilencing device.